Cancer is a term used to describe a group of malignancies that all share the common trait of developing when cells in a part of the body begin to grow out of control. Most cancers form as tumors, but can also manifest in the blood and circulate through other tissues where they grow. Cancer malignancies are most commonly treated with a combination of surgery, chemotherapy, and/or radiation therapy. The type of treatment used to treat a specific cancer depends upon several factors including the type of cancer malignancy and the stage during which it was diagnosed.
Cytoxan is one of the more commonly used cytotoxic agents. This chemotherapeutic agent whose common chemical name is cyclophosphamide has the formula:
cyclophosphamide is a pro-drug for administering the active 4-hydroxycyclophosphamide (HCY) which has the formula:

The compound of Formula I, cyclophosphamide, metabolizes into the compound of formula II-A in the bloodstream when cyclophosphamide is administered to a patient as a therapeutic agent. As active ingredients, the compound of formula II-A exists with its tautomeric form as an aldophosphoramide which has the formula:

Both the compounds of formula II-A and its tautomer, the compound of formula II-B while being the active ingredients are unstable compounds outside the bloodstream. Therefore, in order to administer the compound of formula II-A and II-B to patients, these compounds have to be administered as cyclophosphamide.
Since the compound of formula II-A and its tautomer, the compound of formula II-B are unstable, immunoassays to detect their presence are not practical. In order to determine and/or quantitate, by immunoassays, the presence of the compound of formula II-A and/ors it tautomer, the compound of formula II-B, in the bloodstream of a patient, it has been necessary to trap out the active species, i.e., the compound of formula II-A and II-B. This has been done by protecting the aldehyde group on the active species present in the compound of formula II-B by forming a protected aldehyde such as an oxime or hydrazone. The formation of these protecting groups from the aldehyde group of aldophosphamide can be done by conventional means for forming a protected aldehyde group and the presence of the active ingredients in the bloodstream measured from this stable trapped derivative as described by Ludeman et. al. J. Pharma. Sci., 84(4): PP 393-398, 1995, Zon et. al. J. Pharma. Sci., 71(4): pp 443-446, 1982, and McDonald et. al. Blood, 101(5): pp 2043-2048, 2003. By monitoring the levels of the active cyclophosphamide species in the body and adjusting the dose, the side effects resulting to patients from cyclophosphamide administration can be better controlled and limited. (Ren et. al. Clin. Pharmacol. Ther. 64(3): pp 289-301, 1998, et. al.; Petros et. al., Clin. Cancer Res. 8: pp 698-705, 2002; and Chen et. al. Cancer Research 55: pp 810-815, 1995).
Another reason for monitoring is that there is often high variable relationship between the dose of cyclophosphamide administered and the resulting serum drug concentration which varies the therapeutic effect. The degree of intra- and inter-individual pharmacokinetic variability of cyclophosphamide can be as high as 9-fold (Chang et. al. Pharmacogenetics 7: 211-221, 1997, Ren et. al. Clin. Pharmacol. Ther. 64(3): pp 289-301, 1998) and is impacted by many factors, including:                Organ function        Genetic regulation        Disease state        Age        Drug-drug interaction        Time of drug ingestion        Mode of drug administration, and        Technique-related administration.        
As a result of this variability, equal doses of the same drug in different individuals can result in dramatically different clinical outcomes (Hon et. al. Clinical Chemistry 44, pp 388-400, 1998). The effectiveness of the same cyclophosphamide dosage varies significantly based upon individual drug clearance and the ultimate serum drug concentration in the patient. Therapeutic drug management would provide the clinician with insight on patient variation in both oral and intravenous drug administrations. With therapeutic drug management, drug dosages could be individualized to the patient, and the chances of effectively treating the cancer without the unwanted side effects would be much higher (Nieto, Current Drug Metabolism 2: pp 53-66, 2001).
In addition, therapeutic drug management of cyclophosphamide would serve as an excellent tool to ensure compliance in administering chemotherapy with the actual prescribed dosage and achievement of the effective serum concentration levels. It has been found that variability in serum concentration of the active ingredients of cyclophosphamide is not only due to physiological factors, but can also result from variation in administration technique and ability of the body to absorb and metabolize cyclophosphamide. This is especially true given cyclophosphamide, when administered to a patient, is generally absorbed and metabolized into its active ingredients by the patient at different rates. Therefore, in monitoring the level of these active ingredients in patients by means of an immunoassay, it is important that the immunoassay be able to distinguish the active ingredients of the compound of formula II-A and II-B, from the inactive substance of the compound of formula I, i.e., cyclophosphamide. The problem with antibodies to these active ingredients is that they cross-react with cyclophosphamide making these immunoassays not useful.
Routine therapeutic drug management of cyclophosphamide would require the availability of simple automated tests adaptable to general laboratory equipment. Tests that best fit these criteria are immunoassays. Currently there are no immunoassays for cyclophosphamide administration available and monitoring levels of the active metabolites of cyclophosphamide is conducted by physical methods like high pressure liquid chromatography (HPLC) (Escoriaza et. al. J. of Chromatography B: Biomedical Sciences and applications, 736 (1+2): pp 97-102, 1999). In order to be most effective in monitoring drug levels the antibody should be specific to cyclophosphamide metabolites in their stable form and display very low cross-reactivity to no cross-reactivity to related compounds, particularly cyclophosphamide.